As described in the Official Journal of the European Communities [1998, “Commission Directive 98/86/EC of 11 Nov. 1998,” Official Journal of the European Communities 9.12.98:L334/11], E 410 and E 412 can be differentiated by microscopic examination of their aqueous solutions stained with iodine and potassium iodide. In such preparations, E 410 presents long stretched tubiform cells, separated or slightly interspaced. Their brown contents are much less regularly formed in guar gum than in locust bean gum. E 412 shows close groups of round to pear shaped cells. Their contents are yellow to brown. Thus, theoretically, this microscopy methods should allow identification of guar in mixtures of of guar gum and locust bean gum.
E 410 and E 412, individually or mixed, can be added as food additives, provided that the food label indicates whether only one of these two or a combination of the two additives is present. Both E 410 and E 412 are galactomannans and differences in their chemical composition have been described: galatose:mannose ratios of 1:4 and 1:2, in locust bean gum and guar gum, respectively. Thus, isolated E 410 and E 412 can easily be differentiated from each other by gas-liquid chromatography of the hydrolyzed and derivatized polysaccharides, and after calculation of the galactose:mannose ratio in the resulting chromatogram. However, for these two polysaccharides, and depending of their origin, supplier, and batch, variations in the degree of substitution of the mannan main chain have been described. For example, 37% in E 412 and 23% in E 410 [Cheetham, N. W. H., B. V. McCleary, G. Teng, F. Lum, and Maryanto, 1986, “Gel-permeation studies on xanthan-galactomannan interactions,” Carbohydr. Polym. 6:257-268], galactose:mannose ratios for E 410 and E 412 of 1:2.69 and 1:1.44, respectively [Angelini, E., C. Boniglia, M. Mosca, and G. Bellomonte, 1984, “Detection and determination of plant gums by gas-chromatographic determination of their constituent monosaccharides,” Rivista delta Societa Italiana di Scienza dell'Alimentazione, 13:479–484], or 3.01:1 and 1.48:1, respectively [Preuss, A., and H. P. Their, 1982, “Quantitative analysis of natural thickeners and gums by methanolysis and capillary column gas chromatography,” Z. Lebensm. Unters. Forsch. 175:93–100]. Even galactose:mannose ratios ranging from 3.1:1 to 7.7:1 were determined in locust bean gum fractions solubilized at temperatures between 20° C. and 80° C. [Lopes da Silva, J. A., and M. P. Goncalves, 1990, “Studies on a purification method for locust bean gum by precipitation with ethanol,” Food Hydrocolloids, 4:277–287]. Due to these variations in composition it may be technically impossible in some mixtures of E 410 and E 412 to ascertain the individual content of each gum, and, for the same reasons, a possible adulteration of E 410 with E 412 may be technically very difficult to demonstrate. Cases of adulteration of E 410 with E 412 have been documented. The interest of this type of adulterations can be deduced from the different prizes of the two additives, with E 412 being cheaper than E 410.
Polysaccharides of plant origin are widely used as stabilizing agents in the food industry, and thus many chemical and some physical methods have been used to identify and quantitate these agents in mixtures and foods. None of these methods reviewed by Morley et al. [Morley, R. G., G. O. Phillips, and D. M. Power, 1972, “Fractionation and identification of commercial hydrocolloid stabilising agents,” Analyst, 97:315–319] neither the sequential fractionation scheme described in the cited reference can distinguish E 410 and E 412. More recently, the techniques generally used to identify and quantitate food additives of polysaccharide nature, the same general type of E 410 and E 412, including electrophoresis, gas chromatography and gas-liquid chromatography. None of these methods differentiate E 410 and E 412. Limitations of these methods, particularly when applied to the analysis of foods, are the need of complex extraction prior to analysis and expensive equipment. Furthermore, hydrolysis, the first necessary step for chromatographic analysis of a mixture of E 410 and E 412, will also release, for example, mannose from xanthan, thus affecting the galactose:mannose ratio. Xanthan is another food additive (E 415) which is often used in combination with E 410 and E 412.
In a different described method, E 410, E 412, and other polysaccharides used as food thickeners were isolated, pyrolised, and their products of pyrolysis were analyzed by gas chromatography. Although it was possible to identify a single food thickener by retention index monitoring and selected ion monitoring of the pyrolytic products, accurate analysis of a mixture of food thickeners was not achieved [Sjoeberg, A. M., and H. Pyysalo, 1985, “Identification of food thickeners by monitoring of their pyrolytic products,” J. Chromatography, 319:90–98.].
An assay using the Bandeiraea simplicifolia lectin which allows detection/quantification of guar and locust bean gum in commercial food products has been described [Patel, P. D., and G. B. Hawes, 1988, “Estimation of food-grade galactomannans by enzyme-linked lectin assay,” Food Hydrocolloids, 2:107–118]. Although the referred method detected E 410 and E 412 and not other food additives of polysaccharide nature (xanthan, carrageenan, alginates, and pectin), guar and locust bean gums could not be differentiated.
Finally, DNA-based methods have been described for the detection and/or identification of plants and plant-derived products. For example, in patent WO/9814607, a method is described for detecting a particular plant species in foodstuffs. The method is based in the detection of plant-specific sequences located in the chloroplast DNA. Regions of the chloroplast DNA that contain characteristic sequences of some example plants were identified in the cited patent, allowing the differentiation between oranges and mandarines. Although the methods described in the patent may be applicable to the differentiation of plant species other than those these described in the examples of this patent, these methods were not applied to the differentiation of either guar plant and carob tree neither to their derived products E 410 and E 412, respectively. On the other hand, there are reports of sometimes insufficient phylogenetic resolution in studies based on chloroplastic DNA, i.e., that they do not always allow species identification [Baldwin, B. G., M. J. Sanderson, J. M. Porter, M. F. Wojciechowski, C. S. Campbell, and M. J. Donoghue, 1995, “The ITS region of nuclear ribosomal DNA: a valuable source of evidence on angiosperm phylogeny,” Ann. Missouri Bot. Garden. 82:247–277]. Additionally, as of today, there are not chloroplastic sequences available for the guar plant, and its differentiation from carob tree and other plant species based on these DNA sequences would require first their identification, isolation and sequencing. Finally, for the reasons detailed below, the extraction of DNA, either chlorplastic or not, from thickening agents, may require special methods that are not described in the cited patent.
For the present patent, it is finally relevant the description of a DNA based-method for the detection of wheat contamination in 35 samples of foods and food additives [Allmann, M., U. Candrian, C. Höfelein, and J. Lüthy, 1993, “Polymerase chain reaction (PCR): a possible alternative to immunochemical methods assuring safety and quality of food: Detection of wheat contamination in non-wheat food products,” Z. Lebensm. Unters. Forsch. 196:248–251]. This method is based on the use of PCR and primers specific for the ribosomal DNA, i.e., on sequences of wheat DNA encoding for the RNA components of the ribosomes. It is important for the present patent to note that the authors of the cited method concluded that thickening agents or additives, in particular guar gum and locust bean gum, could not serve as a substrates for DNA isolation, and that the thickening and absorption effects of these additives made DNA extraction impossible.